Project Summary Age-related arterial dysfunction is the main risk factor for cardiovascular diseases (CVD). Recently, we have used short-term and lifelong studies in mice to determine how a Western-style diet (WD; high fat and sugar, low fiber and nutrient density) and aerobic exercise (EX), common lifestyle factors, interact with aging to influence endothelial dysfunction and stiffening of the large elastic arteries. We found that WD accelerates, and EX prevents, these key features of arterial aging via changes in oxidative stress and inflammation. The gut microbiome is a strong modulator of host metabolic health and inflammation that is influenced by age, diet and EX, but there is no information about its effects on arterial function in these or other settings. Our integrative hypothesis is that dysregulation of the gut microbiome (gut dysbiosis) with primary aging and WD consumption, coupled with increased intestinal permeability that allows gut-derived particles to leak into circulation, may act to impair arterial function via changes to adverse gut-derived metabolites such as atherosclerosis-linked trimethylamine-N-oxide (TMAO), and stimulation of toll-like receptor 4-induced pro- inflammatory signaling, whereas chronic aerobic exercise protects against these effects. The purpose of this application is to determine the potential causal role of the gut microbiome in the effects of aging, WD and EX on arterial function, and gain insight into the underlying metabolomic and inflammatory mechanisms. We will employ 3 highly innovative, complementary translational approaches: 1. Mouse studies that allow us to discern cause-and-effect: a) by assessing arterial function with aging WD in the presence vs. absence of the gut microbiome and associated signaling; and b) by determining if vascular phenotypes associated with aging, WD and EX can be transferred via the gut microbiome. We also will explore possible mechanisms using pharmacological inhibition and/or knock-out of suspected pathways. 2. Human studies assessing the time course (temporal associations) of diet (WD vs. non-WD)-induced changes in the gut microbiome vs. arterial function in young and older exercising and non-exercising healthy adults, using a randomized, single-blind, controlled feeding crossover study design. 3. Combined mouse/human studies employing ?humanized? mice to determine if the characteristics present in the human gut microbiome with age, WD and EX predictably influence arterial function. These studies will determine not only changes to gut microbe presence and relative abundance with aging, WD and EX, but also the functional effects of those changes, allowing us to gain novel insight into the role of the gut microbiome in modulating vascular function with aging and these common lifestyle influences. The expected results have the potential to establish the gut microbiome as a key mechanism and therapeutic target for age-related arterial dysfunction, and to identify lifestyle or pharmacological strategies that may preserve microbial health, enhance arterial function and reduce the risk of age-related CVD.